A reductase by micellar cholesterol in CaCo-2 cells

To investigate whether, and by what mechanisms, luminal (dietary) cholesterol regulates cholesterol synthesis in human intestinal cells, HMG-CoA reductase activity, gene expression, synthesis, and degradation were investigated in CaCo-2 cells exposed to taurocholate micelles containing cholesterol. In cells incubated with cholesterol solubilized in 5 mM taurocholate and 30 pM monoolein, HMG-CoA reductase activity was decreased. 25-Hydroxycholestero1, delivered to the cells in the same manner as native cholesterol, was significantly more potent in inhibiting reductase activity and was used, therefore, to investigate mechanisms for sterol regulation. Cells incubated with taurocholate micelles without cholesterol lost cellular cholesterol into the medium causing an increase in HMG-CoA reductase activity and enzyme mass. Although steady-state levels of HMG-CoA reductase mRNA were increased under conditions of cholesterol efflux, synthesis rates of reductase protein were not increased. An increase in activity and enzyme mass in cells incubated with micelles alone, however, was accompanied by a significant decrease in the rate of degradation of reductase protein. In contrast, sterol influx from taurocholate micelles was associated with a marked decrease in HMG-CoA reductase activity and mass without altering mRNA levels except at high concentrations of the polar sterol which did decrease reductase mRNA levels by 50%. The absorption of apical sterol resulted in a significant decrease in the translational efficiency of reductase mRNA and a modest increase in the rate of degradation of the enzyme. Thus, although the primary function of the enterocyte is to transport luminal (dietary) cholesterol to other tissues of the body, apically derived cholesterol enters metabolic pools within the cell which regulates its own cholesterol synthesis. Dietary cholesterol, therefore, will regulate the contribution to the total body cholesterol pool of endogenously derived cholesterol from the intestine. The mechanism for this regulation of intestinal HMGGOA reductase by luminal cholesterol occurs primarily at the post-transcriptional level. -Field, F. J., T. Shreves, D. Fujiwara, S. Murthy, E. Albright, and S. N. Mathur. Regulation of gene expression and synthesis and degradation of 3-hydroxy3-methylglutaryl coenzyme A reductase by micellar cholesterol in CaCo-2 cells. J. Lipid Res. 1991. 32: 1811-1821. Cholesterol requirements of most cells are met by two separate but interrelated processes. One process is the endogenous synthesis of cholesterol. This synthetic pathway, which involves over 20 reactions, is regulated primarily by the activity of HMG-CoA reductase which catalyzes the formation of mevalonate (1, 2 ) . The other process involves the utilization of lipoprotein cholesterol following internalization of the lipoprotein bound to its surface receptor. Under most circumstances, the endogenous synthesis of cholesterol is required only if lipoprotein internalization is insufficient to meet the cholesterol requirements of the cell (3). To maintain cellular cholesterol homeostasis, there exists a rather potent negative feed-back system on the activity of HMG-CoA reductase which results in a decrease in the synthesis of cholesterol if excess sterol enters via the receptor. One mechanism for this feed-back regulation of HMG-CoA reductase activity by sterols is thought to occur at the level of gene expression leading to a decrease in gene transcription (4). Another factor that has been shown to regulate the expression of reductase is controlled degradation of the protein (5-7). The mechanism of this regulation is not well understood but it appears to depend upon the attachment of the enzyme to the endoplasmic reticulum (8, 9). Lastly, in experiments with Chinese hamster ovary cells, there is evidence for a decrease in translation efficiency of mRNA for HMG-CoA reductase resulting in decreased reductase protein and activity (10). In regard to the regulation of cellular cholesterol metabolism, the small intestinal absorptive cell is unique. I n addition to access of cholesterol via the LDL receptor and the biosynthetic pathway, the absorptive cell has acSupplementary key words cholesterol 25-hydroxycholestero1 micelles Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A. 'To whom correspondence should be addressed. Journal of Lipid Research Volume 32, 1991 1811 by gest, on O cber 8, 2017 w w w .j.org D ow nladed fom cess to exogenous luminal cholesterol at its apical membrane. Although it is reasonable to assume that this influx of cholesterol at the apical membrane will contribute to the enterocyte cholesterol pool and result in a decrease in cholesterol synthesis, this remains controversial (11). Results of earlier studies have suggested that luminal bile acids rather than cholesterol regulate cholesterol biosynthesis in the intestine (12). In rat, most dietary studies have shown a lack of response of intestinal cholesterol synthesis to ingested cholesterol (13, 14). These observations have important ramifications as they relate to plasma cholesterol levels and the risk of atherosclerosis. It has been demonstrated that newly synthesized cholesterol from the intestine contributes directly to the plasma pool of cholesterol (15). Since the rate of intestinal cholesterol synthesis is second only to the liver (13), the apparent lack of regulation of endogenously synthesized cholesterol by exogenous cholesterol would result in continued high rates of cholesterol synthesis during times of significant cholesterol influx, a situation that may not be beneficial to the host. This lack of effect of luminal cholesterol on intestinal cholesterol metabolism, however, is species-dependent. Cholesterol synthesis in the intestines of rabbits, guinea pigs, and hamsters appears to be suppressed by dietary cholesterol (16-18). Mechanisms that explain the regulation of HMG-CoA reductase activity by luminal sterols in the intestine in vivo or in vitro (19-21) have not been previously addressed. The present study was undertaken, therefore, to investigate whether luminal sterols regulate HMG-CoA reductase activity in the human intestinal cell line, CaCo-2. The results demonstrate that sterols absorbed from taurocholate micelles by the apical membrane of CaCo-2 cells decrease HMG-CoA reductase mass and activity. A mechanism for this regulation was demonstrated by pulse-chase experiments that showed a decrease in the translational efficiency of HMG-CoA reductase mRNA and an increase in the rate of degradation of the protein.